Sex‐specific repolarization heterogeneity in mouse left ventricle: Optical mapping combined with mathematical modeling predict the contribution of specific ionic currents

Abstract Ventricular repolarization shows notable sex‐specificity, with female sex being associated with longer QT‐intervals in electrocardiography irrespective of the species studied. From a clinical point of view, women are at a greater risk for drug‐induced torsade de pointes and symptomatic long‐QT syndrome. Here, we present an optical mapping (OM) approach to reveal sex‐specific action potential (AP) heterogeneity in a slice preparation of mouse hearts. Left ventricular epicardial repolarization in female versus male mice shows longer and, interindividually, more variable AP duration (APD), yielding a less prominent transmural APD gradient. By combining OM with mathematical modeling, we suggest a significant role of IKto,f and IKur in AP broadening in females. Other transmembrane currents, including INaL, only marginally affect basal APD. As in many cardiac pathophysiologies, increasing [Ca2+]i poses a risk for arrhythmia, the response of AP morphology to enhanced activation of L‐type calcium channels (LTCC) was assessed in a sex‐selective manner. Both APD and its variation increased significantly more in female versus male mice after pharmacological LTCC activation, which we hypothesize to be due to sex‐specific INaL expression based on mathematical modeling. Altogether, we demonstrate a more delayed repolarization of LV epicardium, a leveled LV transmural APD gradient, and a more pronounced epicardial APD response to Ca2+ influx in females versus males. Mathematical modeling quantifies the relative contributions of selected ionic currents to sex‐specific AP morphology under normal and pathophysiological conditions.


| INTRODUCTION
Clinical and experimental studies have revealed sexspecific differences in electrocardiography, particularly with respect to ventricular repolarization properties. At baseline, female sex is associated with longer QT intervals (Abi-Gerges et al., 2004;Locati et al., 1998). Particularly notable become such differences when comparing the prevalence of cardiac arrhythmias between sexes. Women are significantly more prone to symptomatic Long-QT Syndrome (LQTS) and Torsade de Pointes (TdP) tachycardia than men (Ebert et al., 1998;Makkar, 1993). It is widely assumed that differences in ion channel expression and function account for the sex-specific differences in phenotype. However, many of the previous studies on sex-related variations in murine left ventricular ionic current expression and function have reported conflicting findings (Table 1). Crucial repolarizing currents such as the fast transient outward K + current (I Kto,f ) and the ultrarapid delayed-rectifier K + current (I Kur ) have been found mutually up-and down-regulated or not regulated at all in female versus male mice (Brunet et al., 2004;Trépanier-Boulay et al., 2001;Wu & Anderson, 2002). Attempts to interpret such inconsistencies attributed ionic current regulations to varying sex hormone levels. Androgens have been shown to enhance I Kur density and subsequently shorten action potential duration (APD) in male mice (Brouillette et al., 2005), whereas high levels of estrogen may down-regulate I Kto,f and I Kur densities prolonging the APD in female mice (Trépanier-Boulay et al., 2001). Also, 10-12 months (Wu & Anderson, 2002) C57BL/6 septum I K,total (↓) Isolated cardiomyocyte (Patch-clamp, RT-PCR) 10 months (Crump et al., 2016)  2-3 months (Brunet et al., 2004) Abbreviations: I K,total , total outward K + current; I K1 , inward rectifier K + current; I Kss , the steady-state K + current; I Kur , ultrarapid delayed rectifier K + current; I Na , inward Na + current; I NaL, late inward Na + current; I sus , sustained outward K + current; I to,f , fast transient outward K + current; I to,s , slow transient outward K + current.
an elevated late sodium current (I NaL ) component in the AP may increase the susceptibility of female mice to LQTrelated arrhythmias (Wu & Anderson, 2002). Whether the increased I NaL function, however, simply reflects a decreased repolarization reserve due to down-regulation of K + channels remains elusive. The majority of experimental research in this field exploited models of dissociated cardiomyocytes, particularly useful for combining single-cell electrophysiology with molecular analysis. However, cell isolation entails noteworthy drawbacks. Not only will the regional origin of the studied cells get at least partially lost, but also will enzymatic digestion harm the extracellular matrix and hence important ion channel modulators or even accessory subunits (Kline & Mohler, 2014;Liu & Melchert, 2010;Salvage et al., 2020). In the present study, we therefore established an optical mapping approach to visualize transmembrane voltage in thin slices of murine ventricular tissue offering accurate locoregional AP features. We sought out to investigate the APD and its regional intra-and interindividual variability in the left ventricle of adult male and female mice, both under baseline conditions and in response to pharmacological calcium channel activation resembling a cardiac stress factor. Mathematical modeling approximated the respective contribution of selected ionic currents on AP morphology.

| Animals
All animal experiments comply with the EU Directive 2010/63/EU on the protection of animals used for scientific purposes. Studies were approved by the local Animal Care and Use Committee of the University of Düsseldorf (# O7/11) and the Animal Ethics Committee of the North Rhine-Westphalia Nature, Environment and Consumer Protection Agency (LANUV). Adult male and female C57BL/6J mice (20-30 g; 22-27 weeks; Janvier Labs), maintained at a 12/12 h light/dark cycle and fed ad libitum water and chow diet were used for the study. All mouse experiments were performed in the same time window of the day (±2 h) to control for putative heterogeneities in cardiac electropysiology caused by circardian rhythm.
After dissecting atria and major vessels, ventricles were embedded in 4% low-melt agarose (Art. No. 6351.2, Carl Roth) dissolved in phosphate buffered saline at 37°C. The agarose blocks were chilled quickly until complete solidification. The agarose-embedded ventricles were glued with tissue adhesive histoacryl (175,182, Bbraun) on a specimen holder and were sectioned transversally into 350 μm thick slices employing a vibratome (LEICA VT1200S) with steel blades at a progression speed of 0.03 mm/s and lateral blade vibration amplitude of 2 mm (Wang et al., 2015). During the sectioning process, the tissues were kept in an ice-cold oxygenated bicarbonatebuffered extracellular solution (95% O 2 , 5% CO 2 ) containing 10 μM (−)-blebbistatin.
Immediately after sectioning, the slice was transferred into a custom-made circulation chamber filled with 10 μM (−)-blebbistatin containing bicarbonate-buffered solution. By positioning a glass spiral before the chamber entrance and regulating the solution flow rate, the temperature of the chamber was kept at 35°C. The slices were stimulated using a unipolar platinum-iridium electrode (ThermoFisher) connected to an electrical stimulator (STG4002, Multi Channel Systems MCS GmbH) with a cycle length of 200 ms, a pulse width of 1 ms, and 1.5 times of threshold strength (3-5 V). For the cardiac slices to reach an electrophysiological steady state, they had to be continuously paced for at least 30 min. During this time, the signal-to-noise ratio of fluorescence intensity likewise increased and reached a stable state. All short-axis slices included in this study were taken from the middle one third of the heart to control for AP heterogeneity over the longitudinal axis (base/apex).

| Optical mapping
To excite Di-8-ANNEPS, an LED light source (LEX3-G; 525 nm; SciMedia/Brainvision) connected to a THT Macroscope (SciMedia/Brainvision) was used. The light passed through an excitation filter (BP531 nm/40) was deflected by a dichroic mirror (580-FDI) towards the perfusion chamber and focused onto the slice. The fluorescent signals emitted by the slice were gathered via a 1.0X objective (LEICA) and filtered using a longpass filter (LP600 nm). The filtered fluorescent emission was captured by a MiCAM05-N256 imaging system (SciMedia/Brainvision) equipped with a CMOS image sensor (mapping field area: 10 × 10 mm (256 × 256 pixels); frame rates: 1 kHz; SciMedia/Brainvision). The effective spatial resolution is 50 μM/pixel. The proprietary BV Workbench (Brainvision) was used to operate the optical recordings and to store them in the appropriate data format before being submitted to customized MATLAB analysis.

| FPL64176 application
After APD recordings had reached a steady-state, FPL64176 (250 nM) was added to the circulating extracellular solution, while APs were continuously monitored in time intervals of 5 min until FPL64176-induced prolongation of APD again reached steady-state.

| Analysis of optical data
Data processing, analysis, and visualization were carried out applying self-developed MATLAB scripts. Processing of optical data comprised ensemble averaging, drift correction by polynomial fitting, and spatial mean filters of 3 × 3 pixels. The data processing was partially inspired by RHYTHM source code (Gloschat et al., 2018). The data were analyzed by extracting activation times and AP duration at 30%, 50%, 80%, and 90% of repolarization (APD 30 , APD 50 , APD 80, and APD 90 ). In order to investigate APDs, we adjusted the "findpeaks" function to identify local maxima and widths at various peak amplitude levels. The vector drawing program Inkscape was used to rearrange and edit MATLAB plots and maps.

| Mathematical modeling
Simulations were created using a dynamic mathematical model of a mouse ventricular AP (Li et al., 2010). The predominant transmembrane currents, ionic pumps, and exchangers are included into the model. Also, intracellular Ca 2+ homeostasis formulations are taken into account. OpenCARP environment has been used to implement the simulations (Plank et al., 2021). The AP traces were resampled to 1 ms time intervals resembling the optical mapping temporal resolution.

| Statistical analysis
Data were reported as mean ± standard deviation, and statistical analyses using Student's t-tests determined statistical significance reported as: *p < 0.05, **p < 0.01, and ***p < 0.001. When several slices from an individual animal were investigated, hierarchical, nested statistics were applied to test for significant differences (Eisner, 2021;Sikkel et al., 2017). Coefficient of variability statistics was used to estimate the spread of APDs and its gradients in males versus females, and the signed-likelihood ratio test (SLRT) was used to examine their significance (Krishnamoorthy & Lee, 2014). We used the R package "cvequality" to test for significant differences.

| Sex-specific repolarization properties in mouse heart slices
Cardiac slice preparations have been developed for systematic optical analyses of regional gradients of transmembrane voltage and [Ca 2+ ] i across the ventricles (Ripplinger, 2018;Wang et al., 2015;Wen et al., 2018). First, we optimized the optical system and sample preparation, increasing both temporal and spatial resolution and signal quality (see Methods). For optical analysis of locoregional AP features in adult male and female mice, we focused on three parameters of interest within the LV: (i) the whole LV free wall, (ii) epicardium (outer one third of the LV free wall), and (iii) the LV transmural gradient of APD (Figure 1a). Color-coded maps reveal a sex-specific spatial distribution of APD at 90% repolarization (APD 90 ) across the LV at a stimulation frequency of 5 Hz ( Figure 1b). As quantified in Figure 1c, epicardial APD 90 was significantly greater in LV slices of females than of males (p = 0.0061, n = 6 male and n = 9 female mice, respectively), whereas neither the overall LV APD 90 nor their standard deviations (SD) differed between sexes. Instead, a strong negative transmural gradient of APD 90 towards epicardium was observed in slices of male mice, which was significantly reduced in slices of female mice ( Figure 1d; Table S1, p = 0.00002). In addition, analyses of coefficients of variations (COV) showed significantly increased dispersion of epicardial APD 90 (MSLRT = 5.1, p = 0.023) and of transmural APD gradients (MSLRT = 18.4, p = 1.7 × 10 −5 ) in female versus male mice.
With the most robust sex-specific APD heterogeneity apparently occurring in epicardium, we focused our further investigation specifically on this area of interest. Overlaying epicardial AP morphologies disguises longer APD, particularly at late repolarization, in female over male mice, as depicted and quantified in Figure 1e. For quality control, it should be mentioned that the absolute APD 90 in male LV was 69 ± 3 ms, which is in perfect agreement with the values recorded from intact mouse heart at this pacing rate (Table 2) (Knollmann et al., 2001;Saito et al., 2005).

| Simulation of ionic current contribution to the mouse cardiomyocyte AP morphology
Mathematical models have been an essential tool for understanding and exploring the complex interactions of ionic currents and their contribution to AP generation and morphology. The evident controversy on sex-specific ion channel expression and function (Table 1) prompted us to exploit mathematical modeling in order to contrast and compare the effects of putative differential ionic current modulation in more detail. Figure 2a shows a schematic diagram of an electrophysiological model of a single F I G U R E 1 Optical mapping allows for sex-specific comparison of the left ventricular transmural mouse action potential. (a) Schematic illustration of the areas of interest analyzed in the left ventricle (LV). An appropriate angle was chosen to exclude anterior and inferior heart regions. Whole LV free wall, epicardium (one third of the LV free wall), and the transmural gradient of action potential duration (APD) were investigated parameters. Papillary muscles were explicitly excluded from LV wall analysis. (b) Optical mapping of APD at 90% repolarization (APD 90 ) in a representative male (left panel) and female (right panel) LV short-axis heart slice, respectively. (c) Bar graphs showing APD 90 and its standard deviations (SD), calculated from whole LV free wall (LV) and epicardium (epi) of slices from male (M) and female (F) mice (M: n = 6, F: n = 9; **p = 0.0061; # MSLRT = 5.1, p = 0.023). (d) Quantification of transmural gradient vectors of APD 90 (M: n = 6, F: n = 9; ***p = 0.000018; ### MSLRT = 18.4, p = 1.7 × 10 −5 ) (e) Overlay of male and female epicardial APs (regions defined in b by dashed lines), respectively, and quantification of the APD at indicated % repolarization (n = 6 slices from N = 6 male mice, n = 12 slices from N = 9 female mice; APD 80 ; **p = 0.009). mouse cardiomyocyte established and further optimized by the Bondarenko group (Bondarenko, 2004). We have implemented the formulations in openCARP (Plank et al., 2021). As most debates on sex-specific ion channel regulation in cardiomyocytes are about potassium channels, we first compared the maximum conductivities of potassium currents implemented in the model (Figure 2a, lower panel). It shows that in the mouse cardiomyocytes, g Kr and g Ks play rather minor roles compared to others, for which we excluded them from further analysis. Instead, the influence of the remaining K + currents on AP morphology was assessed in more detail by mathematically simulating respective increases or decreases in current densities of up to ±50%. A stimulus pulse was applied for a period of time of 5 s at a frequency of 5 Hz. We focused on changes in AP morphology induced by the trains of simulation while conductivity varied and overlayed the respective last APs of two stimulations to better appreciate the differences ( Figure 2b). As expected, a reduction in all K + current densities prolonged the APD and solely changes in I K1 , altered the resting membrane potential. As the latter, however, did not show any reliable sex-specific difference in previous experimental data (suppl. Figure 1), we further excluded I K1 from investigation in this context. A decrease in I Kto was predicted to prolong APD 30 and APD 80 by up to 16% and 28%, respectively. For a reduction in I Kur , the model suggested an increase in APD up to 4% and 29% at early and late repolarization phases, respectively. Finally, for decreases in I Kss , the model indicated APD changes below 4%. These findings are summarized in Figure 2b (right panel), plotting APD 30 and APD 80 as a function of respective conductivity (g): Whereas both I Kto and I Kur have a similarly strong impact on late repolarization (APD 80 ), early repolarization (APD 30 ) is predominantly shaped by only I Kto . Based on literature (Lowe et al., 2012), we finally aimed at simulating also the effects of the basal late sodium current on AP morphology. Given the Markov model for I Na depicted in Figure 2b, the rate constant of channel transition from a fast-inactivated state (IF Na ) to the open state (O Na ) was modulated by ±50%. The model yielded a rather moderate increase in APD 80 by 9% upon a 50% increase in the rate of re-opening. Altogether, the mathematical simulations point to a predominant influence of I Kto on early repolarization, whereas both I Kto and I Kur share a similar and dominant contribution to late repolarization, partially counteracted by basal I NaL .

| L-type calcium channel (LTCC) activation disguises sex-dependent repolarization reserve
Female sex has been recognized as an independent risk factor for drug-induced LQTS (Li et al., 2013  mishandling and resulting signaling events in cardiomyocytes, referred to as the Ca 2+ vicious cycle, link a number of cardiac pathophysiologies with cardiac arrhythmias including LQTS (Hegyi et al., 2021), with increased Ca 2+ influx prolonging the electrographic QT interval (Giudicessi & Ackerman, 2016). The pharmacological LTCC agonist FPL 64176 is a convenient tool to potentiate the voltagedependent calcium current, which balances AP repolarization (Fan & Palade, 2002), and hence afforded us to assess APD morphology under Ca 2+ stress conditions in a sex-dependent manner. As shown in Figure 3a, APD 80 mapping of a representative LV epicardial region indicated that FPL 64176 (250 nM) delayed late AP repolarization in heart slice preparations from both female and male mice, stimulated at 5 Hz. However, detailed quantification of APDs after incubation with FPL 64176 demonstrates significant prolongation of medium and late repolarization by 30%-80% only in female sex, whereas there was only a tendency for a repolarization delay in male sex (Figure 3b). APD at 30% repolarization remained unaffected, irrespective of sex. These data indicate a sexdependent repolarization reserve becoming evident by LTCC activation.

| Simulation indicates a dominant role for I Na-L in shaping AP repolarization under calcium stress
Finally, we tried to gain more mechanistic insight into the experimentally observed sex-dependent differences in repolarization reserve by mathematical modeling.  Single mouse cardiomyocyte APs were simulated while shifting voltage-dependence of Ca v channel activation to more negative potentials (−15 mV), well mimicking the action of FPL 64176. The individual role of potassium (I Kto , I Kur ) and sodium currents (I NaL ), which had been reported to be differentially regulated in a sex-specific manner and which were identified to contribute significantly to late AP morphology in our modeling experiments above, were assessed by adapting their respective current densities. First, for an arbitrary increase in APD 80 , the corresponding extent of current modulation was calculated. A reduction of I Kto by 23%, of I Kur by 21%, and an increase in I NaL by 52%, was each sufficient to yield a prolongation in APD 80 of 10% under model baseline conditions. As shown in Figure 4a, shifting voltage-dependence of Ca v channel activation by −15 mV resulted in a maximum increase in APD 80 by 40%, which was not further affected by the additional reductions in I Kto or I Kur calculated above. However, the calculated increase in I NaL conductivity of 52%, yielding a 10% prolongation of APD 80 at regular Ca v channel function, more than doubled APD 80 when challenged by Ca v channel overactivation. In Figure 4b, the putative current contributions to late repolarization behavior in response to a gain of Ca v channel function are summarized. Taken together, mathematical modeling indicates a prominent role of I NaL in shaping the late AP morphology under calcium stress.

| DISCUSSION
Sex-specific differences in cardiac AP morphology due to differential ion channel regulation are widely accepted. However, most studies on sex-dependent expression and function of cardiac ion channels have been performed in dissociated cardiomyocytes, which do not provide the spatial context that is, on the one hand, necessary to perform correct comparisons between males and females and, on the other hand, may also contribute to functionally relevant heterogeneity between sexes (Antzelevitch & Fish, 2001;Bartos et al., 2015). Here, we demonstrate optical mapping of membrane voltage in a slice preparation of mouse heart as a feasible tool to reveal sex-specific AP heterogeneity at high spatial resolution. By combining this approach with mathematical modeling, we were able to generate hypotheses how the observed heterogeneity in AP morphology might relate to respective single current densities in cardiomyocytes. The present study reveals marked sex-specific differences in AP repolarization in epicardial regions of the LV, with longer APD at late repolarization in female than in male mice. Thus, the transmural gradient in late APD observed in male mice becomes leveled in female mice, which demonstrates sex-specific patterns of transmural heterogeneity. These findings are in good agreement with electrographic studies in humans demonstrating a larger T peak -T end interval in men than in women (Smetana F I G U R E 3 Sex-specific increase in action potential duration upon L-type calcium channel activation. (a) Optical mapping of APD 80 in a representative region of the left ventricle (LV) before and after the application of the pharmacological calcium channel activator FPL 64176 (250 nM) in male (upper) and female (lower) heart slice preparations. Two representative APs averaged from indicated areas before and after application of FPL 64176 are overlaid (right). (b) Overlay of male and female LV APs (before and after FPL application) and quantification of APD at indicated % of repolarization (n = 5 slices of N = 5 male mice, n = 5 slices of N = 5 female mice; APD 50 : p = 0.00077, APD 80 : p = 0.00623, APD 90 : p = 0.00899). Color codes for male (black), female (red), and after FPL 64176 application (blue), respectively. et al., 2003). In addition, the present study highlights a strikingly higher degree of interindividual AP and APD gradient variability in female mice that may be attributed to menstrual stages, which were not controlled herein. Also, structural heterogeneity by lipid or collagen deposition may add to both intra-and interindividually varying ion channel expression patterns (Gerdts & Regitz-Zagrosek, 2019). Particularly, influencing factors like hormonal state and age have always fueled the discussion about the sex-specificity of the cardiac AP (Brouillette et al., 2003;Brouillette et al., 2005;Brunet et al., 2004;Lowe et al., 2012;Saito et al., 2009;Trépanier-Boulay et al., 2001;Wu & Anderson, 2002;Yusifov et al., 2021). It is also noteworthy to mention that there is considerable variance between reported APD parameters in isolated cells ( Figure S1, Table 2), which most likely reflects uncontrolled methodological and spatial determinants. By preserving a much more physiological cell composite in the heart slice preparation, our study convincingly confirms both longer epicardial APD and increased APD variance in female than in male mice.
The cardiac AP results from a complex voltagedependent interplay of a number of ionic currents deand repolarizing the resting membrane potential. Thus, changes in AP morphology will reflect changes in underlying ionic currents. During recent years, extensive studies on isolated cardiomyocytes have come up with genetic or functional regulation of several candidate currents, in an attempt to explain the different features of the cardiac AP in male versus female mice; however, with numerous discrepancies in the literature. For instance, there exist several reports indicating a reduction in the ultrarapid delayed-rectifier potassium current I Kur in female mouse ventricles, which would comprehensibly prolong the APD (Brunet et al., 2004;Crump et al., 2016;Saito et al., 2009;Trépanier-Boulay et al., 2001), whereas other studies described I Kur as either indifferent from or even larger than in male mice (Brouillette et al., 2003;Brouillette et al., 2005;Wu & Anderson, 2002). As mentioned above, parameters such as the sex hormonal state or spatial context may well account for the diverging reports on sex-dependent I Kur expression. More robust, there is evidence for a smaller density of the transient outward potassium current I to in females (Brunet et al., 2004;Crump et al., 2016;Saito et al., 2009); the underlying ion channel alpha-subunits K v 4.2 and K V 4.3 have been shown to be less expressed in females versus males. It should be kept in mind that most ion channels assemble as multi-protein complexes with their function being not only determined by gene or protein expression of the pore-lining subunits, but also by auxiliary subunits and numerous post-translational modifications affecting channel numbers on the cell surface or their gating properties . Intriguingly, the beta-subunit KCNE4 modulates both I Kur and I to functions F I G U R E 4 Simulation of sex-specific contribution of ionic currents on action potential duration upon L-type calcium channel activation. (a) Simulation of APs upon a gradual shift in voltage-dependence of I CaL activation: voltage of half-maximum activation (V 1/2 ) was shifted to negative potentials by up to 15 mV at four different conditions: control (male sex); I Kto (−23%): 23% reduction in g Kto ; I Kur (−21%): 21% reduction in g Kur ; I NaL (+52%), 52% increase in rate constant (k, Figure 2c). Note the striking delay and heterogeneity in late repolarization, particularly under increasing I NaL . (b) Calculations of APD 80 upon the indicated shift in voltage-dependence of activation of I CaL under corresponding sex-specific current modifications. (c) APD 80 alteration under indicated sex-specific current modification with (gray) and without (white) a 15 mV shift in V 1/2 of I CaL to negative potentials. (Crump et al., 2016). Among the sex-specific ionic currents may also be the late sodium current I NaL , which has been shown to particularly increase in female compared to male mice when specifically activated (Lowe et al., 2012). Using mathematical modeling of mouse cardiomyocyte electrophysiology, we were able to demonstrate the quantitative contribution of each of the predominant ionic currents to AP morphology. Under physiological conditions, we hypothesize I to to shape both early and late repolarization, while I Kur and-to a lesser extent-I NaL affect only late repolarization. In contrast, the currents I Kss , I Kr , and I Ks are unlikely to substantially contribute to sex-specific AP morphology in mice, as their densities are rather negligible. As not being considered in the model, it should be kept in mind, that post-translational regulation of these currents may indeed change their contribution (Morotti et al., 2014;Tapa et al., 2020).
We finally sought to evaluate the sex-specific AP morphology in response to modified Ca 2+ flux into cardiomyocytes. In a number of cardiac pathophysiologies, including hypertrophy and Long-QT syndromes, increased Ca 2+ influx plays a central role (Antoons et al., 2007;Hennessey et al., 2014;Wemhöner et al., 2015). Often a shift in voltage-dependent activation of I CaL to more negative potentials is observed, increasing I CaL window current, which prolongs the APD and promotes the formation of early after depolarizations (Song et al., 2015). Here, we have shown that the response in AP morphology to pharmacological activation of I CaL is sex-specific, with a much more pronounced APD prolongation in females. Obviously, differential expression of I CaL density may account for such observation. In female rats and dogs, cardiomyocytes exhibit higher Ca 2+ channel densities and hence greater I Ca than in cardiomyocytes from respective male animals (Saito et al., 2009;Vizgirda et al., 2002;Xiao et al., 2006). However, for mice, Johnson et al. showed that only decreased levels of estrogen are associated with an increased number of cardiac LTCCs (Johnson et al., 1997). Based on our simulation experiments increasing window I CaL , we assume an outstanding role for I NaL in shaping the sex-specific response of AP morphology. Among the above specified determinants of female APD prolongation in mice, I NaL undoubtedly outstands in delaying APD 80 in response to enhanced Ca 2+ influx. Being aware of important differences in the spatial expression of cardiac ion channels between mice and humans, it suggests itself that a more prominent role of I NaL in drug-induced LQTS, which occurs more often in women than in men (Li et al., 2013), should be considered. It is worth mentioning that besides ion channels, also ion transporters may be expressed in a sex-specific manner and hence influence the sex-specific cardiac electrophysiology (for review: [James et al., 2007;Yang & Clancy, 2012]). Thus, at high intracellular Ca 2+ concentrations and subsequent formation of early afterdepolarizations (EADs), a possible role of the sodium/calcium exchanger NCX has to be taken into account (Parks & Howlett, 2013).
Our study has limitations that are worth highlighting. The currently available mathematical models of single mouse ventricular myocytes show significantly shorter APDs than the ones recorded from cardiac tissue. Lacking mechanical load or mechanical uncoupling agents may contribute to this difference. Continuously revising the models based on experimental data hence appears not only necessary but will improve our quantitative understanding of sex-specific differences in cardiac electrophysiology, eventually giving rise to sex-specific models much better adopted to given medical needs. We selected specifically the LV epicardial region for our study, because of the significant amount of published data on sex-specific ion channel regulation in cardiomyocytes isolated from this area and because of the availability of a respective mathematical model. Nevertheless, the herein introduced experimental approach may be generalized to other cardiac regions, including the septum and RV. Transmural slices at high spatial resolution provide even the tool to investigate sex-specific intra-heart APD heterogeneity. Such analyses may open new doors to understand the sex-specific differences in cardiac arrhythmia susceptibility. Despite the present limitation of focussing on sex-specific characterization of membrane voltage in healthy mouse heart slices, the approach is well suited to gain also comprehensive insight into sex-specific differences in regional remodeling processes (e.g., membrane voltage and calcium signaling) after myocardial infarction and in hypertrophy. On a perspective, both superresolution microscopic and spatial omics technologies may well be combined with the herein established optical mapping and mathematical modeling approach to help test unbiased hypotheses of regulated ion channel function in both physiological and pathophysiological conditions.

AUTHOR CONTRIBUTIONS
S. Erfan Moussavi-Torshizi, Ehsan Amin, and Nikolaj Klöcker conceived the project. S. Erfan Moussavi-Torshizi and Ehsan Amin performed the experiments and analyzed the data. Ehsan Amin and Nikolaj Klöcker wrote the manuscript.

ACKNOWLEDGMENTS
The study was supported by the Deutsche Forschungsgemeinschaft (DFG, grant: CRC 1116, TPA01 to N.K.). The authors thank Stefan Schaetz and Thomas Becher for their technical support. Open Access funding enabled and organized by Projekt DEAL.